A novel microfluidic platform to study exosome biology in PAH.

一种用于研究多环芳烃外泌体生物学的新型微流体平台。

• 批准号: 10158068
• 负责人: VINICIO A DE JESUS PEREZ
• 金额: $ 23.64万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-25 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158068
• 关键词: Acoustics; Address; Animal Model; Antibodies; Bioinformatics; Biological; Biological Markers; Biology; Blood Vessels; CD81 gene; Cardiovascular system; Cell Culture Techniques; Cells; Characteristics; Chronic; Culture Media; Cultured Cells; Data Set; Disease; Endothelial Cells; Endothelium; Etiology; Experimental Models; Exposure to; Genetic; Goals; Heart failure; Inflammation; Inflammatory; Injury; Lab-On-A-Chips; Life; Lung; Machine Learning; Mediating; Methods; Microfluidics; Molecular; Molecular Genetics; Nucleic Acids; Outcome; Pathologic; Pathologic Processes; Pattern; Phase; Phenotype; Physiological; Physiological Processes; Population; Process; Production; Proteomics; Reporting; Reproducibility; Resolution; Role; Seeds; Signal Transduction; Sorting - Cell Movement; Stress; Surface; Techniques; Technology; Testing; Therapeutic Agents; Time; Vascular remodeling; Vision; angiogenesis; base; cytokine; design; endothelial dysfunction; exosome; extracellular vesicles; hemodynamics; machine learning algorithm; magnetic beads; monolayer; multiple omics; new technology; novel; novel therapeutics; pressure; protein metabolite; pulmonary arterial hypertension; response; shear stress; stressor; technological innovation; tool; transcriptome sequencing

The endothelium is the cellular monolayer that covers the inner lining of the entire circulatory system. Endothelial dysfunction is a feature of pulmonary arterial hypertension (PAH), a life-threatening disease associated with abnormally high pulmonary pressures and chronic right heart failure. Due to the limitations of available static cell culture and animal models, our understanding of the mechanisms that orchestrate the initiation and perseverance of endothelial dysfunction in PAH remains incomplete. Given that endothelial dysfunction is a common finding in PAH, an understanding of the mechanism behind maladaptive endothelial responses could help accelerate the discovery of novel therapies for PAH. Presently, it is believed that endothelial derived exosomes contribute to PAH by carrying signals that trigger maladaptive endothelial responses in the setting of injury. Exosomes are cell-derived small (~30-150 nm) extracellular vesicles that carry proteins, metabolites and nucleic acids involved in a variety of physiological and pathological processes. While it is known that exosomes carry molecular and genetic factors associated with angiogenesis, inflammation and vasoreactivity, a comprehensive assessment of exosome cargo of healthy and dysfunctional PMVECs has been hindered by current low-yield exosome isolation techniques. These techniques cannot perform real-time dynamic exosome isolation from pulmonary microvascular endothelial cells (PMVECs) exposed to PAH-associated stressors. To address this unmet need, we have designed the MFES (Multifunctional Exosome Sorter) that can dissect the whole exosome population into subpopulations based on size and surface markers. MFES is the first lab-on-a-chip platform that integrates: 1) a vessel-on-a-chip module for real-time characterization of PMVEC functional responses across a wide range of physiological and pathological parameters, 2) a module for high-yield exosome size-based isolation, 3) a surface marker based exosome sorting using magnetic beads, and 4) multi-omics phenotyping of exosomes of PMVECs. Here, we are proposing a technology that can enable broadly to investigate the two main defining characteristics of exosomal subtypes, i.e., size and surface markers, both separately independently, and in combination sequentially. We will characterize changes in exosome cargo in healthy and PAH PMVECs exposed to shear stress-related conditions in the MFES. We will isolate subpopulations of exosomes based on size and surface markers and characterize them for their cargo (Aim 1). Then, we will determine whether exosomes derived from stressed PMVECs can induce pathological changes in healthy PMVECs cultured in a microfluidic culture chip (Aim 2). This technological innovation enables to study endothelial exosome biology in a setting that represents the flow dynamics associated with PAH. Further, the use of cutting-edge -omics technologies, bioinformatic analysis integrated with machine learning algorithms to analyze the purified exosomes is expected to yield a comprehensive dataset of exosome cargo profiles and open exciting opportunities for investigating the biological role of exosomes in PAH pathobiology and the testing of novel therapeutic agents.

内皮是覆盖整个循环系统内壁的细胞单层。内皮 功能障碍是肺动脉高压（PAH）的特征，这是一种威胁生命的疾病 异常高的肺部压力和慢性右心力衰竭。由于可用静态单元的局限性 文化和动物模型，我们对策划启动和毅力的机制的理解 PAH中的内皮功能障碍仍然不完整。鉴于内皮功能障碍是常见的发现 PAH，对不良适应性内皮反应背后机制的理解可以帮助加速 发现PAH的新型疗法。目前，人们认为内皮派生的外泌体有助于 PAH通过携带触发受伤情况下不良适应性内皮反应的信号。外泌体是 细胞来源的小（〜30-150 nm）细胞外囊泡，含有蛋白质，代谢物和核酸 在各种生理和病理过程中。虽然众所周知，外泌体携带分子和 与血管生成，炎症和血管反应性相关的遗传因素，对 当前的低收益外泌体隔离阻碍了健康和功能障碍PMVEC的外部货物 技术。这些技术无法从肺部执行实时动态外泌体隔离 暴露于PAH相关的应激源的微血管内皮细胞（PMVEC）。为了满足这种未满足的需求， 我们设计了可以剖析整个外泌体种群的MFE（多功能外泌体分散器） 基于大小和表面标记的亚群。 MFES是第一个集成的实验室平台： 1）芯片上的容器，用于实时表征PMVEC功能响应的实时表征 生理和病理参数，2）用于高产外泌体大小隔离的模块，3）a 基于表面标记的外泌体分类使用磁珠，4）外泌体的多词表型 PMVEC。在这里，我们提出了一项技术，可以广泛调查两个主要定义 外泌体亚型的特征，即大小和表面标记，均单独独立，并在 顺序组合。我们将表征健康和PAH PMVEC中外泌体货物的变化 在MFE中剪切应力相关的条件。我们将根据大小和 表面标记并为其货物描述它们（AIM 1）。然后，我们将确定外泌体是否 源自应力的PMVEC可以诱导在微流体中培养的健康PMVEC中的病理变化 文化芯片（目标2）。这种技术创新使得在一个环境中研究内皮外泌体生物学 表示与PAH相关的流动动力学。此外，使用尖端 - 理论技术， 与机器学习算法集成的生物信息学分析预计可以分析纯化的外泌体 为了产生外部货物概况的全面数据集，并开放了令人兴奋的机会来调查 外泌体在PAH病理生物学和新型治疗剂的测试中的生物学作用。